Ehrlichia chaffeensis causes human monocytic ehrlichiosis (HME), an emerging tick-borne zoonosis. From the site of infected tick bite on human skin, E. chaffeensis infects monocytes and spreads via the bloodstream to various tissues, causing a systemic febrile disease. HME is characterized by fever, headache, myalgia, thrombocytopenia, leucopenia, and elevated liver-enzyme levels, but complications such as pulmonary insufficiency, renal failure, encephalopathy, and disseminated intravascular coagulation can cause death [Paddock C D, et al. (2003) Clin Microbiol Rev 16: 37-64]. Early diagnosis and the proper treatment with doxycycline are critical to prevent complications. The disease is of particular threat to the immunocompromised and the elderly people [Paddock C D, et al. (2003) Clin Microbiol Rev 16: 37-64].
E. chaffeensis is a small obligatory intracellular bacterium. It belongs to the family Anaplasmataceae in the order Rickettsiales that includes many understudied pathogens of veterinary and public health importance [Rikihisa Y (2010) Nat Rev Microbiol 8: 328-339]. By electron microscopy, E. chaffeensis is a polymorphic bacterium (0.2-2.0 μm in diameter), and can be morphologically categorized as small dense-cored cells (DCs) or large reticulate cells (RCs) [Popov V L, et al. (1995) J Med Microbiol 43: 411-421]. DCs are approximately 0.2-0.5 μm in diameter, which is close to the size of the elementary body of Chlamydia and larger viruses such as Vaccinia virus. By light microscopy, it is not possible to distinguish individual RCs and DCs, since E. chaffeensis aggregates inside eukaryotic host cells. The characteristic clump of intracellular E. chaffeensis organisms is termed as “morula” (mulberry in Latin) [Rikihisa Y (2010) Nat Rev Microbiol 8: 328-339]. However, when they are freshly isolated from host cells and dispersed, smaller bacteria (<0.5 μm) are more densely stained with basic dye than larger bacteria (>0.5 μm); therefore, they were defined as DCs and RCs, respectively [Zhang J Z, et al. (2007) Cell Microbiol 9: 610-618]. DCs are more resistant to strong sonication and more infectious than RCs [Cheng Z, et al. (2008) J Bacteriol 190: 2096-2105]. In cell culture, a biphasic developmental cycle has been reported: initially small infectious DCs bind to and internalize into host cells, which then develop into larger replicating RCs inside a membrane-lined compartment that resembles early endosomes. After replication in expanding inclusions, the mature RCs transform back into DCs prior to release from the host cells [Zhang J Z, et al. (2007) Cell Microbiol 9: 610-618; Cheng Z, et al. (2008) J Bacteriol 190: 2096-2105]. In patients' blood specimens, monocytes were primarily infected with E. chaffeensis, and hence, the disease was named as “monocytic ehrlichiosis” to distinguish it from “granulocytic ehrlichiosis” caused by infection with granulocyte-tropic Ehrlichia sp. [Paddock C D, et al. (2003) Clin Microbiol Rev 16: 37-64]. E. chaffeensis can replicate well in several mammalian cell lines including canine histiocytic leukemia (DH82), human acute leukemia (THP-1), human promyelocytic leukemia (HL-60), human embryonic kidney (HEK293), and monkey endothelial (RF/6A) cells [Mott J, et al. (1999) Infect Immun 67: 1368-1378; Liu H, et al. (2012) Cell Microbiol 14: 1037-1050; Miura K, et al. (2011) Infect Immun 79: 4947-4956].
Entry into the permissive eukaryotic host cells is essential for E. chaffeensis to sustain its life, since its small genome of 1.18 Mb lacks a large portion of metabolic genes that are required for free living [Dunning Hotopp J C, et al. (2006) PLoS Genet 2:e21]. E. chaffeensis also lacks LPS, peptidoglycan, lipoteichoic acid, and flagella that engage Toll-like or NOD-like receptors, or scavenger receptors [Rikihisa Y (2010) Nat Rev Microbiol 8: 328-339; Rikihisa Y (2010) Nat Rev Microbiol 8: 328-339]. E. chaffeensis entry and subsequent infection of THP-1 cells, but not binding are almost completely inhibited by monodansylcadaverine (MDC), a transglutaminase inhibitor [Lin M, et al. (2002) Infect Immun 70: 889-898]. MDC is known to block Neorickettsia risticii (formerly Ehrlichia risticii) entry and infection of P388D1 cells, vesicular stomatitis virus uptake and receptor-mediated endocytosis of α2-macroglobulin by Swiss 3T3 mouse cells, but not the uptake of latex beads by P388D1 mouse macrophages [Levitzki A, et al. (1980) Proc Natl Acad Sci USA 77: 2706-2710; Messick J B, et al. (1993) Infect Immun 61: 3803-3810; Schlegel R, et al. (1982) Proc Natl Acad Sci USA 79:2291-2295]. E. chaffeensis entry into THP-1 cells, leading to productive infection, is dependent on the host-cell surface lipid rafts and glycosylphosphatidyl inositol (GPI)-anchored proteins [Lin M, Rikihisa Y (2003) Cell Microbiol 5: 809-820]. Furthermore, lipid raft-associated protein caveolin-1, but not clathrin localizes to the E. chaffeensis entry site [Lin M, Rikihisa Y (2003) Cell Microbiol 5: 809-820]. After entry, E. chaffeensis replicates in the membrane-bound compartment resembling an early endosome as it contains early endosome antigen 1 (EEA1), Rab5, and transferrin receptor [Mott J, et al. (1999) Infect Immun 67: 1368-1378]. Several intracellular bacteria are known to enter host cells by using their specific surface protein collectively called as ‘invasin’ or ‘internalin’ [Pizarro-Cerda J, et al. (2006) Cell 124: 715-727]. However, detailed mechanisms of E. chaffeensis entry were unknown; particularly regarding the involvement of any specific bacterial surface protein that can function as an invasin and its cognitive host-cell receptor [Rikihisa Y (2010) Nat Rev Microbiol 8: 328-339].
The importance of E. canis as a veterinary pathogen in conjunction with the recent identification of E. chaffeensis as the cause of an emerging tick-borne zoonosis has highlighted the need for improved diagnostics and vaccines for both veterinary and human ehrlichioses, and thus the need for identification of immunoreactive proteins.